PROJECT SUMMARY ? PROJECT 4 The ultimate goal of this proposal is to understand the role of innate immunity within the context of oncolytic herpes simplex viral (oHSV) therapy for glioblastoma (GBM), a highly fatal brain tumor. oHSV treatment of GBM relies on cancer-specific replication of the virus leading to tumor destruction with minimal toxicity to adjacent non-neoplastic tissue. Its safety in patients has been proven, yet evidence for significant efficacy remains to be established. Project 4 will focus on the host's natural killer (NK) cell response following oHSV administration. Due to their strong antiviral properties, NK cells represent a potential barrier to oHSV therapy. Alternatively, the anti-tumor NK cell response has the potential of augmenting the tumor clearing properties of oHSV therapy. Our previously published studies demonstrated that: 1) NK cells rapidly respond oHSV and eliminate the virus before it can effectively replicate and disseminate throughout the GBM; 2) transient immune modulation delaying the NK cell response to oHSV, even with a single injection of TGF-beta, significantly enhances oHSV replication, spread and anti-tumor efficacy following inoculation. As a result, we hypothesize that NK cells coordinate a robust inflammatory response following initial oHSV administration that limits oHSV replication, spread, and tumor lysis, thereby creating a barrier to effective oHSV therapy for GBM. A corollary to this hypothesis is that by understanding how NK cells recognize oHSV-infected GBM we can best modulate this process so as to optimize this highly selective therapy for GBM. We therefore recently developed a novel assay whereby we cloned each HSV1 gene into a vector that allowed us to determine its ability to either induce or inhibit NK cell activation. We discovered the mechanism by which NK cells recognize and destroy HSV-infected targets prior to the development of a primary immune response. We term this fundamental, novel discovery passive antibody dependent cellular cytotoxicity or passive ADCC, whereby the Fc fragment of IgG (IgGFc) forms a bridge between the NK cell Fc receptor for IgGFc, called CD16, and the GBM cell expressing HSV Us8, which encodes the Fc binding protein glycoprotein E (gE). The CD16-IgGFc-gE ternary complex activates NK cells to destroy oHSV-infected GBM without the requirement for the antigen-specific Fab portion of IgG. In this proposal, Project 4 will: (1) further investigate passive ADCC to best understand how to modulate the process so as to enhance the efficacy of oHSV therapy for GBM with Project 1; (2) determine the role of TGF-? signaling in regulating the NK cell interaction with oHSV-encoded viral proteins, specifically as relates to passive ADCC and to NOTCH signaling with Project 3; (3) To assess the NK cell immune response in patients with recurrent GBM who will be receiving our oHSV, rQNestin34.5 in Project 2. By elucidating how NK cells recognize and destroy oHSV- infected tumors in the context of other pro- and anti-inflammatory processes, we will best understand how to optimize this neuro-selective treatment for GBM as well as discern the key mechanistic signals leading to NK cell-mediated clearance of viral infection.